Chromatin condensation and DNA fragmentation are hallmarks of apoptotic cell death. During the past few years, two nuclear effectors of apoptosis, the DNA Fragmentation Factor (DFF) or Caspase-Activated Deoxyribonuclease (CAD), and Apoptosis Inducing Factor (AIF), have been identified. Extensive biochemical and cellular studies have established that these factors play major roles in executing the characteristic changes in nuclear morphology and integrity during apoptosis. [unreadable] [unreadable] AIF has been implicated as a caspase-independent factor, which upon release from the mitochondria, translocates to the nucleus and causes initial chromatin condensation and large fragment DNA cleavage. Targeted deletion of AIF abolishes the first wave of apoptosis indispensable for early embryonic morphogenesis, while the presence of AIF in extra-mitochondrial space is sufficient to kill cells. AIF appears to be a bifunctional protein with a resident mitochondrial function and an apoptogenic function. [unreadable] [unreadable] DFF is a molecular complex consisting of DFF40 (CAD), a novel endonuclease, and two splicing variants of its inhibitor, DFF45 (ICAD-L) and DFF35 (ICAD-S). During apoptosis, DFF45 gets cleaved by caspases, resulting in the release and activation of DFF40, which produces advanced chromatin condensation and oligo-nucleosomal DNA fragmentation. [unreadable] [unreadable] In this application, we propose to use a combination of X-ray crystallography, biochemistry and cell biology to elucidate the molecular basis of AIF and DFF function.